Quantification of "Uncultivated" Species in Periodontal Biofilms

About 50% of the species present in subgingival biofilms are as yet uncultivated. The role of these species in periodontal pathogenesis is unknown.

AIMS: to develop a high throughput method to detect and quantify a wide range of cultivated and uncultivated species that may be associated with periodontal disease or health.

METHODS: Oligonucleotides targeting the 16S rRNA gene were designed and synthesized. Target species included those associated with periodontal health and disease, such as Streptococcus gordonii, Fusobacterium nucleatum sp polymorphum Porphyromonas gingivalis, Prevotella intermedia, Actinomyces gerencseriae and Aggregatibacter actinomycetemcomitans. Sequences for uncultivated species, such as TM7 sp AH040, Selenomonas sp CS002, Haemophilus sp BJ095 and Desulfobulbus sp R004 were also synthesized. Sequences were labeled with digoxigenin. Five serial dilutions from pure cultures were prepared and laid onto a nylon membrane. 10 ng of extracted total nucleic acids from each species were used as targets. The same procedure was employed for bacterial mixtures, supragingival and subgingival plaque samples. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Direct hybridization was performed in a checkerboard format. Chemiluminescent signals were visualized after film exposure. In a clinical pilot study, subgingival plaque samples from periodontally healthy and periodontitis subjects were examined.

RESULTS: Probes provided specific signals. Probe sensitivity reached 10⁵ cells for most species. Extracted nucleic acids provided signals at the 10 ng level. F. nuc. ss polymorphum and A. gerencseriae were the most abundant cultivated species in clinical samples. Among uncultivated species, Selenomonas sp CS002 and Prevotella sp DO0027 were the most numerous. P. gingivalis and Desulfobulbus sp R004 were only detected in periodontitis patients.

CONCLUSIONS: Direct hybridization of total nucleic acids using oligonucleotide probes permits quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.